KR100261881B1 - High pullulan content products, and its preparation and uses - Google Patents

Abstract

PURPOSE: To produce a pullulan concentrate industrially advantageously at a high productivity by continuously culturing a culture medium having a high sugar content while keeping the viscosity of the medium at a specified low value, and to establish the use of the concentrate. CONSTITUTION: A microorganism capable of producing pullulan is continuously cultured in a culture medium contg. 10-20w/v% sugar with the viscosity of the medium kept at 30 cP, giving a concentrate of pullulan having an average mol.wt. lower than 250,000. The concentrate is used as a thickener, a coating material, an adhesive, a molded article, etc., in the fields of food and beverage, cosmetics, medicines, materials for agriculture and stock raising, materials for paper manufacture, materials for mining industry, etc.

Description

High content of pullulan and its manufacturing method and uses

The present invention relates to a high content of pullulan and a method and a use thereof, and more particularly to containing a microorganism having a pullulan production ability and containing a sugar concentration of 10 to 20 w / v%. The present invention relates to a high content of pullulan having an average molecular weight of 250,000 or less obtained by continuous culture while maintaining the viscosity of the medium in a nutrient medium at 30 centipoise (hereinafter, simply referred to as “cp”) or less, and a method and a use thereof. .

Pullulan is industrially produced as viscous glucan obtained by aerobic culture of microorganisms having pullulan production ability in a nutrient medium containing monosaccharides and oligosaccharides.

As the production method, for example, as disclosed in Japanese Patent Application Laid-Open No. 49-42894, a method of increasing the productivity of pullulan by adjusting the initial pH of the nutrient medium to 5.5 to 8.0, and particularly adjusting the high pH, As disclosed in Japanese Patent Application Laid-Open No. 52-130993, a method of increasing the productivity of pullulan has been proposed by culturing microorganisms having pullulan production ability in the presence of amylase inhibitory substances while preventing the viscosity of the nutrient medium from decreasing. . However, it has been found that all of these methods are batchwise, i.e., not necessarily satisfactory in terms of industrial production of pullulan.

Meanwhile, a method for producing pullulan by continuously culturing microorganisms having pullulan generating ability is disclosed in the following publication.

(1) Bulmaer et al., Applied Microbiology and Biotechnology, Vol. 25, pp. 362-365, 1987.

(2) Boa et al., Biotechnology and Bioengineering, Vol. 30, pp. 463-470, 1987.

(3) McNeil et al., Biotechnology and Bioengineering, Vol. 33, pp. 1210-1212, 1989.

(4) McNeil et al., Enzyme and Microbial Technology, Vol. 12, pp. 521-525, 1990.

However, these publications have not been biochemically examined for the production of pullulan by continuous culture, and the concentration of sugars such as glucose, sucrose, peat hydrolyzate, and the like is low, as low as 2 to 5 w / v%. In addition, the viscosity of the nutrient medium has not been examined. The pH of the nutrient medium is not clear from Boa et al., But is maintained at pH 5.5 in Bulmer et al., And McNeil et al. Reports that pH 4.5 is a suitable pH. The highest value of pullulan productivity (gL ^-1 h ^-1 ) (hereinafter referred to simply as "pulullan productivity" in the present invention) per 1 L of culture medium and 1 hour of culture is the lowest value of 0.48 described in the literature of Boa et al. The period of the continuous culture is also unknown. Thus, it is extremely difficult to use these reports to produce high content of pullulan which is beneficial for industrial production.

It is desired to solve the above-mentioned drawbacks of the conventional method and to establish a method for producing a high content of pullulan by a novel continuous culture method which is industrially advantageous and the use thereof.

The inventors of the present invention have made a method for producing a high content of pullulan by continuous culture, in particular, using a nutrient medium containing a high concentration of saccharides, and a high content of pullulan which is advantageous for industrial production by paying attention to the viscosity of the culture solution and the productivity of the pullulan. Eager research was continued with the aim of establishing the production method and use thereof.

As a result, unlike the conventional art, pullulan having an average molecular weight of 250,000 or less obtained by continuous culture of microorganisms having pullulan production ability in a nutrient medium containing a sugar concentration of 10 to 20 w / v% at a viscosity of 30 cps or less is obtained. The relics have been found to have high pullulan productivity by continuous culture and to be stable over a long period of time, and furthermore, that industrial production is extremely advantageous. Therefore, it contains a manufacturing method by continuous culture and a high content of pullulan to be produced, and is used in various fields such as thickeners, coating agents, adhesives, moldings, foods, cosmetics, pharmaceuticals, agriculture, forestry and fisheries materials, paper processing materials, and light industry materials. It was established to complete the present invention.

Hereinafter, the present invention will be described in more detail.

The microorganism having a pullulan generating ability used in the present invention may be any strain as long as it is a strain capable of producing pullulan. For example, Aureobasidium fermentans var fermentans IFO 6410, Aureobasidium fermentans var fusca IFO 6402, Aureobasidium pullulans Microorganisms belonging to the genus Aureobasidium, such as IFO 6353 and Aureobasidium pulluans IFO 4464, or mutants thereof can be suitably used.

As the nutrient medium, ones containing a carbon source, a nitrogen source, an organic nutrient source, an inorganic substance, and the like appropriately are used. As the carbon source, for example, glucose, maltose, maltooligosaccharide, isomaltoligosaccharide, starch syrup or starch hydrolyzate, sucrose, fructose, saccharified starch, invert sugar, isomerized sugar, molasses, or the like, may be selected. The concentration is 10 to 20 w / v%, more preferably 10 to 18 w / v%. As the nitrogen source, one or two or more selected from inorganic nitrogen sources such as ammonium salts and nitrate salts or organic nitrogen sources such as glutamate salts, peptone, yeast extract and corn steep liquor are used. As other inorganic substances, phosphate salts, magnesium salts, iron salts and the like are suitably used.

The culture used in the present invention is carried out aerobic while maintaining the viscosity of the nutrient medium to 30 cp or less in a continuous method. Preferably, the culture is carried out under a condition selected from a range of about 25 to 30 ° C. at a pH of 4.0 or less, more preferably pH 2.0 or more and 4.0 or less.

In the method of maintaining the viscosity of the nutrient medium used in the method of the present invention at 30 cps or less, for example, the dilution rate of the nutrient medium is changed by the feeding rate of the nutrient medium which is additionally supplied while discharging a part of the nutrient medium intermittently or continuously. An enzyme that partially hydrolyzes amylases, such as liquefied α-amylase or isoamylase, which is usually adjusted in the range of 0.01 to 0.1 h- ¹ or partially hydrolyzes pullulan to cause a decrease in viscosity of the nutrient medium. It can add and implement an appropriate amount. Moreover, if necessary, it is also advantageous to use a precultured nutrient medium.

The high pullulan-containing culture solution thus obtained is sterilized and concentrated in accordance with a conventional method, and decolorized and desalted as necessary, and the high pullulan content in solution or powdered again to recover powdered high pullulan content. The high content of pullulan produced in this way contains pullulan in the range of about 50 w / w% or more, preferably about 55 to 90 w / w% or more, per solid, and its average molecular weight is usually 300,000 or less, preferably Is in the range of about 40,000 to 250,000. If necessary, the product may be made into higher purity pullulan by molecular weight fractionation, vessel solvent precipitation separation, or the like.

The high content of pullulan of the present invention is water-soluble, thickening, film-forming, gloss, transparency, gas-barrier, oil-resistant, flame-resistant, adhesive, moldability, edible Since it has characteristics such as feeding) and indigestion, it can be advantageously used as a thickener, a coating agent, an adhesive, a molded article, or the like as its use.

In addition, the high content of pullulan of the present invention is a food (eg, Bifidobacterium microbial growth promoter, dietary fiber food, low-calorie foods, etc.), cosmetics (eg dentifrice, latex, Creams, packs, hair dressings, shampoos, bathing agents, etc.], medicines (eg, tablets, capsules, substitute plasma, etc.), agricultural and forest shrinkage materials [eg, coating seeds, granulation (造粒)] Pesticides, molding fertilizers, molding fillers, etc.], paper processing materials [e.g., sizing agents, sizing agents], mining industry materials [e.g. wastewater treatment agents, welding rods, molds, etc.] have.

When used in various applications for the high content of pullulan of the present invention, the high content of pullulan of the present invention may be contained alone or the high content of pullulan of the present invention, together with glycerin, sorbitol, maltitol, and lactitol, depending on the purpose. Polyhydric alcohols, such as these, can also be contained advantageously.

If desired, the high content of pullulan of the present invention and other materials, such as polysaccharides, plasticizers, extenders, excipients, fillers, thickeners, surfactants, foaming agents, antifoaming agents, pH adjusters, stabilizers, One or two or more selected from flame retardants, mold release agents, antibacterial agents, coloring agents, flavoring agents, nutrients, preferences, flavoring agents, medicinal substances, and bioactive substances may be appropriately contained. The high content of high content of pullulan of the present invention may be sufficient to include the high content of pullulan in the process until the manufacture of the intended product is completed. Examples of the method include mixing, kneading, dissolving, dipping, applying, spreading, Well-known methods, such as spraying and injection, are selected suitably.

Specifically, for example, in the case of thickener production, in the case of coating agent production, the method disclosed in Japanese Patent Application Laid-Open No. 51-51543, Patent Publication No. 52-41252, Patent Publication No. 58-213076, etc. is used. Japanese Patent Publication No. 52-56603, Patent Publication No. 53-19416, Patent Publication No. 53-119741, etc. are used, and in the case of adhesive production, Japanese Patent Publication No. 51-119321, Patent Publication So-53-54593, Patent Publication No. 54-108828 and the like can be used. In the case of molding production, Japanese Patent Publication No. 50-116545, Patent Publication No. 51-4272, Patent Publication No. 51-100470, etc. are used, and in the case of food production, Japanese Patent Publication is disclosed. Japanese Patent Laid-Open No. 50-6441, Japanese Patent Laid-Open No. 63-28369 in the case of cosmetics production, using the method disclosed in Japanese Patent Application Laid-Open No. 53-79045, Patent Publication No. 60-219238, and Patent Publication No. The method disclosed in Japanese Patent Laid-Open No. 53-12417, Japanese Patent Laid-Open No. 57-67602, and Japanese Patent Laid-Open No. 63-122739 can be used in the case of manufacturing a pharmaceutical.

Hereinafter, a method for producing a high content of pullulan by continuous culture of the present invention will be described according to the following experiment.

[Experiment 1]

[Influence of Sugar Concentration and Viscosity Control on Pullulan Productivity]

[Experiment 1-A]

[Viscosity Control by Feeding Nutritional Medium]

The nutrient medium shown in Table 1 was placed in an ego jar fermentor, autoclaved at 120 ° C. for 20 minutes and cooled to about 27 ° C. according to a conventional method, and then the microbial seed culture liquid was sterile. After incubation, the mixture was incubated with aeration and stirring while adding hydrochloric acid or sodium hydroxide to maintain a pH of 3.6 to 3.8.

As the culture medium, aerobic incubation of Oreovacidium pullulanse IFO 6353, a strain of a nutrient medium having the same composition, was used for about 24 hours at about 27 ° C.

TABLE 1

The viscosity control method of the nutrient medium was carried out by adjusting the dilution rate to further supply the nutrient medium while discharging the nutrient medium intermittently or continuously. The feeding of the nutrient medium was started after the nutrient medium reached steady state. In measuring the viscosity of the nutrient medium, a part of the nutrient medium was sampled during continuous culture to remove the bacteria, and then the viscosity of the sample was measured using a rotary E-type viscometer (30 ° C).

The measurement of pullulan productivity was calculated | required by measuring about (g) of pullulan per 1 L of nutrient medium and 1 hour of culture with respect to the nutrient medium sample collected between 300-320 hours of continuous cultivation. The term "amount of pullulan" as used herein refers to the amount of polymeric material that is insoluble in 75 v / v% methanol and hydrolyzed by pullulanase (EC 3.2.1.41) to produce mainly maltotriose. It means.

As a control, the same continuous culture was performed under conditions without additional supply of nutrient medium and viscosity control.

The results are shown in Table 2.

TABLE 2

Note: In the table, "cp" means centipoise.

As is apparent from the results in Table 2, when the nutrient medium was discharged from the nutrient medium containing various concentrations of saccharide, the nutrient medium was additionally supplied, and the viscosity thereof was controlled to continuously culture the saccharide medium to 10-20 w / v%, It has been found that high productivity of 0.88 gL ⁻¹ h ⁻¹ or more can be obtained when the viscosity of the nutrient medium is preferably 30 cps or less in the range of 10 to 18 w / v%.

[Experiment 1-B]

[Viscosity Control by Isoamylase Coexistence]

Using a nutrient medium having the same composition as in Experiment 1-A, the nutrient medium was additionally supplied while the nutrient medium was discharged so as to control the viscosity of the nutrient medium to 60 cps or more and 90 cps or less, and further, isoamylase ( By incorporating LinHo Biochemistry Research Institute), it was maintained in the range of pH 2.6 to 3.0 while maintaining in the various viscosity ranges shown in Table 3. Other conditions were carried out similarly to experiment 1-A.

The results are shown in Table 3.

TABLE 3

Note: In the table, "cp" means centipoise.

As apparent from the results of Table 3, when the continuous culture was carried out while controlling the viscosity of the nutrient medium by isoamylase addition, the nutrient was in the range of 10 to 20 w / v%, preferably 10 to 18 w / v%. It has been found that extremely high pullulan productivity of at least 0.92 gL-1h-1 can be obtained when the viscosity of the medium is maintained at 30 cps or less.

[Experiment 2]

[Influence of pH Control on Pullulan Productivity in Viscosity-Controlled Continuous Cultures]

Pullulan productivity was investigated by continuously culturing at various pHs using a nutrient medium having a sugar concentration of 16 w / v% and maintaining the viscosity of the nutrient medium below 30 cps in the presence of isoamylase as in Experiment 1-B.

The results are shown in Table 4.

TABLE 4

As apparent from the results of Table 4, when the pH control in continuous culture is set to pH 4.0 or lower, preferably pH 2.0 or higher and 4.0 or lower, extremely high pullulan productivity of 0.92 gL ^-1 h ^-1 can be obtained. It turned out.

In Example A below, the manufacture example of a high pullulan content, and Example B demonstrate the use example of a high pullulan content, respectively.

Example A-1

[Pululan high content]

Except for using high DE starch syrup (trademark of SUN ROSE, Japan), 14 w / v%, oreobacidium pullul as a saccharide. The seed culture solution of Lance IFO 6365 was inoculated and cultured continuously. While discharging the culture medium, the nutrient medium was further supplied at a dilution rate of 0.01 to 0.04 h ^-1 while maintaining the viscosity of the nutrient medium below 30 cps and the pH in the range of 3.6 to 3.8. It was. The nutrient medium was recovered from the 4th day after the start of the culture, and bactericidal decolorization, concentration and drying were carried out according to a conventional method to obtain a high powdery pullulan content.

The analysis results of this product are shown in Table 5.

TABLE 5

This product can be used for thickeners, coating agents, adhesives, moldings, and food, cosmetics, pharmaceuticals, agriculture, forestry and fishery materials, paper processing materials and mining materials.

Example A-2

[Pululan high content]

A seed culture solution of oreovacidium pullulan IFO 6353 was inoculated in a nutrient medium composed of the same ingredients as in Example A-1, followed by continuous culture. While discharging the culture solution, the nutrient medium was further supplied at a dilution rate of 0.01 to 0.02 h ⁻¹ to add liquefied α-amylase (trade name, Hi-Maltosin S, sold in Japan). By maintaining the viscosity to 30cp or less and the pH was maintained at 3.2 ~ 3.4 and continuous culture for 2 months at 27 ℃. In the same manner as in Example A-1, the nutrient medium was recovered and treated to obtain a powdery high content of pullulan.

The analysis results of this product are shown in Table 6.

TABLE 6

This product can be used for thickeners, coating agents, adhesives, moldings, and food, cosmetics, pharmaceuticals, agriculture, forestry and fishery materials, paper processing materials and mining materials.

Example A-3

[Pululan high content]

Except for the use of maltose-containing starch syrup (trademark sold in Japan, the trademark "MALTO-RUP") as a sugar, the concentration 11, 12, 13, 14, 15, 16 or 18 w / v%. Continuous culture was inoculated with a seed culture solution of oreovacidium pullulan IFO 4464 in a nutrient medium having the same composition as 1. The nutrient medium was kept at 30 cps or lower by adding isoamylase (sold by Japan Institute for Biochemistry) while supplying the nutrient medium at a dilution rate of 0.01 to 0.02 h ^-1 while discharging the culture. And the pH was maintained at 2.4, 2.5, 2.6 or 2.8 and continuous culture was carried out for 2 months at 27 ℃. The nutrient medium was recovered in the same manner as in Example A-1, and the membrane was filtered and concentrated according to a conventional method to obtain a high content of liquid pullulan.

The analysis results of these products are shown in Table 7.

TABLE 7

These products can be advantageously used in thickeners, coating agents, adhesives, moldings and the like, and in food, cosmetics, pharmaceuticals, agricultural and forestry fisheries materials, paper processing materials, and mining materials.

Example B-1

[Sliding contact part sealing composition]

Glycosyl sucrose was added to 56 parts by weight of glycerin and 6 parts by weight of powdered pullulan high content prepared by the method of Example A-1, and then stirred and mixed while heating to 110 ° C. to sell glycosyl sucrose. , 137 parts by weight of the trademark "COUPLING SUGAR") was added, followed by degassing and stirring while preventing evaporation of water.

The viscosity of this composition was about 300 cp. When the composition was used as a sealing composition for the sliding contact portion of an aerosol container with a gas, in particular a liquefied gas, as a propellant, the composition was left at 45 ° C. for 6 months and at 1 year at -5 ° C., but the gas barrier performance was abnormal. It showed satisfactory performance without.

In addition, since the composition is non-toxic and harmless to humans and families, it is advantageous as a sliding contact sealant of an aerosol container for filling various household goods, foodstuffs, cosmetics, medicines, etc., in which gas or liquefied gas with a pressure difference is encountered in the sliding contact part. It is available.

Example B-2

[Pickled radish]

Pickle 30 kg of radishes according to the usual method and continue to pickle with 10 g saccharin, then 1.7 kg of bran, 10 g saccharin, 30 g glycine, 200 g sodium glutamate, 70 g complex seasoning, 500 g sorbitol, 360 g saline, shochu (35) The pickling was carried out in a mixture of 340 g of powdery pullulan high content prepared by the method of 250 ml and A-2.

This product had almost no viscosity decrease even after 12 days of manufacture, and the bran was well adhered to the product, so that both gloss and texture were good.

Example B-3

[Insulating material]

16 weight part of powdery pullulan high content manufactured by the method of Example A-1 was melt | dissolved by heat-dissolving 16 weight part of glycerin and 68 weight part of water, and the cooling material was manufactured by mixing and packing an appropriate amount of antimicrobial agent here.

Cooling this product has a high cooling effect, so it can be used advantageously to cool the head when a person has a fever or to maintain the freshness of plants such as vegetables.

Example B-4

[Coat membrane]

Using a liquid pullulan high content prepared by the method of No. 4 of Example A-3 in a concentration of 1.0 w / w% aqueous solution, it was immersed in fresh eggs within 10 hours after laying for 30 seconds and then 30 ℃ It dried at the temperature of 2 hours, and formed the coating film on the egg shell surface.

The eggs thus obtained were stored at room temperature (15-25 ° C.), and their edible period was compared with the control untreated eggs. As a result, the shelf life of the column in which the coating film was formed was extended about 5 to 10 times further.

Example B-5

[Extraction packing material]

In a small package for black tea made of commercially available filter paper having a relatively large mesh, a high content of liquid pullulan prepared by the method of No. 6 of Example A-3 was used as a 10 w / w% aqueous solution. This was coated by the Calender method and dried by hot air at 60 ° C.

As much as one time the amount of black tea was added to the prepared small package and sealed by heat seal. A commercially available small package containing black tea of the same quality was used as a control. The relative humidity was maintained at 60% and the temperature at 30 ° C., and left for one month, followed by pouring hot water into it to compare the packages as the superiority of the extract obtained by extracting black tea.

Coated with a high content of pullulan of the present invention was also extracted easily as a control when pouring hot water. All extracts were transparent. However, it was excellent in coating with high pullulan in any point of aroma, color, and taste of the extract.

Example B-6

[Adhesive paste for cardboard]

A liquid high content of pullulan prepared by the method of No. 5 of Example A-3 was used as an aqueous solution of 3 w / w% concentration. 10 parts by weight of 10 w / v% sodium hydroxide was added to 100 parts by weight of 20 minutes for 20 minutes. It stirred and set it as the carrier part. Separately, 40 parts by weight of corn starch was added to 100 parts by weight of water to make a slurry, and then 1 part by weight of borax was added to prepare a main part. The carrier part was gradually added and mixed in this main part, and it stirred for 5 minutes, and manufactured the flavor.

This paste has a smaller viscosity change than the conventional starch paste, and is bonded to the B grade liner 240 g / m2 and the semi-central 125 g / m2 using a double-sided corrugator. As a result of the bonding test, the bonding starch showed a tendency to cause poor bonding at 120 m / min or more in the conventional starch paste, whereas bonding was possible without problems even at 160 m / min when the paste of the present invention was used. It was also satisfactory.

Example B-7

[Solid adhesive]

A mixture of 30 parts by weight of dimethyl sulfoxide, 25 parts by weight of water, 5 parts by weight of erucinan, 5 parts by weight of powdered pullulan high content prepared by the method of Example A-2 and 2 parts by weight of benzylidene xylitol was prepared. After stirring for 1 hour at 90 ° C. to dissolve, the resultant was poured into a lipstick-type container having a cylindrical, repeatable vertical movement of 14 mm in diameter and 50 mm in height, and cooled at room temperature to prepare a solid adhesive. .

The adhesive was applied to kraft paper, which resulted in a thin, uniform coating and sufficient initial adhesion. This adhesive had a relatively small change in hardness with temperature and extremely good applicability and adhesion.

Example B-8

[film]

A high liquid pullulan content prepared by the method of No. 4 of Example A-3 was used as an aqueous solution having a concentration of 15 w / w%, where 1 w / w% of carrageenan and 0.1 w / w of sucrose monolaurate per solid. After dissolving% and pouring this liquid on the polyester film, it was made into a film with a thickness of 0.03 mm with a pulling-out speed of 3 m / min, and it dried by hot air of 90 degreeC, and it was made into the product.

Unlike pullulan alone film, this product does not dissolve rapidly in aqueous system, but slowly dissolves and disintegrates and can be eaten.

Therefore, it can be advantageously used as a packaging agent such as a powdery medicine or the like which is difficult to eat as a medical wafer, and also as a film for fixing an artificial tooth since it has adhesiveness when dissolved and disintegrated.

Example B-9

[fiber]

A liquid pullulan high content prepared by the method of No. 1 of Example A-3 was used as a 40 w / w% solution, and the alginic acid was dissolved therein so as to have a concentration of 2 w / w% per solid. The resulting solution was made into a spinning stock solution, which was 60 캜, and extruded into a room temperature air at a pressure of 3 kg / cm 2 from a cylindrical nozzle of 0.3 mm in diameter and 1 mm in length to form a strand, followed by moisture Was wound up in a winder while evaporating to dryness.

The fiber produced had a thickness of about 20 μm and had sufficient strength, which can be processed into arithmetic, knitting, weaving, and the like. Moreover, since it is characterized by being hydrophilic, non-toxic, and non-irritating to the skin, it can be suitably used for cotton wool, menstrual cotton, gauze, surgical thread, and the like.

When mixed with other fibers, the hygroscopicity, non-electrostatic property, and dyeability can be utilized, and they can also be used as underwear or other cloth.

Example B-10

[paper]

A mixture of fibers of high pullulan content prepared by the method of Example B-9 was cut into 5 to 10 cm, and a mixture of half the amount of wood pulp of this fiber was prepared, which was about 50 times the amount of this mixture. The mixture was uniformly suspended in 75 v / v% methanol of to treat in a paper machine.

The drying roll was adjusted to 50-80 ° C., and dried, and then lightly passed through the calendar roll to prepare paper.

This paper has a smooth surface, relatively low gloss, and is similar to the paper of Japanese paper. It blended well with writing ink and was free of ink stains.

This paper is easily soluble in hot water, making it suitable for special paper such as secret documents. In addition, since it can be eaten, it is suitable also as a food packaging material, such as a medicine packaging, seasoning, coffee, and cocoa.

Example B-11

[Foaming sheet]

60 parts by weight of dioctyl phthalate was added as a plasticizer to 100 parts by weight of polyvinyl chloride resin, and 30% by weight of a 50 w / w% aqueous solution of a high content of powdered pullulan prepared by the method of Example A-2 was added to the mixture. After adding w / w%, the mixture was sufficiently mixed in a mixer, and then used an applicator to form a sheet of 3 mm thickness on an aluminum plate, which was heat-treated in a hot stove at 190 ° C. for 10 minutes to obtain a foaming ratio of about 5 Foam sheets with uniform cells were prepared.

This product is suitable for soundproofing materials, insulation materials, packaging containers, cushioning materials, etc. When this product was immersed in river water and left to disintegrate within one month, the high pullulan-free content of the present invention remained intact even after 12 months.

Example B-12

[Golf tee]

10 parts by weight of powdered pullulan high content prepared by the method of Example A-2 and 4 parts by weight of acidic clay were sprayed while stirring to obtain a water content of about 30 w / w%, which was carried out at an injection molding machine at 120 ° C. After molding, the product was immersed in a shellac-alcohol solution and air dried.

The product breaks up into small chunks by shots and then decays slowly by rainwater to biodegrade.

Therefore, this product does not harm the aesthetics of the golf course and does not destroy the environment.

Example B-13

[Flowerpot]

A mixture of 100 parts by weight of the high content of powdered pullulan prepared by the method of Example A-1, 15 parts by weight of glycerin and 5 parts by weight of sorbitol was molded using an injection molding machine at 135 ° C., immersed in molten wax, and then cooled. The product was obtained.

The product is suitable for transplant pots because it is slowly decaying and biodegradable. Plants grown in these pots can be transplanted without pulling them out of the pots, so there is no risk of damage to the plants.

Example B-14

[capsule]

40 parts by weight of the high powdery pullulan content prepared by the method of Example A-1, 60 parts by weight of gelatin and 30 parts by weight of glycerin were mixed, 80 parts by weight of water was added thereto, heated to dissolve at 70 ° C. and degassed to prepare a solution for coating. Then, this was used to prepare a soft capsule containing the contents of high vitamin E oil in a conventional manner.

Unlike gelatine-only capsules, this product has a high gas barrier property, not only keeps vitamin E stable, but also dissolves rapidly in aqueous systems.

Example B-15

[Membrane fertilizer]

Powdered pullulan high content prepared by the compounding fertilizer (N = 14 w / w%, P ₂ O ₅ = 8 w / w%, K ₂ O = 12 w / w%) and the method of Example A-1, The ratios of calcium sulfate and water are 70, 10, 15, and 5, respectively, by weight, and the mixture is sufficiently mixed, and then heated to 80 ° C. in an extruder [L / D ratio = 20, compression ratio = 1.8, diameter of die = 30 mm]. To obtain the title product.

This product does not require fertilizer containers, is easy to handle, has a strength suitable for whole-layer fertilization, and further, it is possible to control the dissolution rate of fertilizer components by changing the mixing ratio. If necessary, the rod-like fertilizer can be easily mixed with plant hormones, agricultural chemicals, soil improving agents and the like.

Example B-16

[Tobacco moldings]

To 50 parts by weight of the powdered tobacco raw material prepared from the pale yellow tobacco plant, 200 parts by weight of a 2 w / w% aqueous solution of a high content of powdered pullulan prepared by the method of Example A-2 and 0.1 parts by weight of lactitol were mixed, and 0.2 mm 65 parts by weight of sheet-like tobacco having a moisture of 13 w / w% was obtained by injecting onto a stainless-steel endless belt from a slit and infrared drying.

This product is suitable as a cigarette or filler for cigarettes and a binder for cigars and cigarilro. In addition, this product suppresses the deterioration of various components of tobacco and has a high degree of preservation, and does not generate odor and odor during smoking, and thus has a good aroma and taste. It was. By varying the blending ratio of pullulan in this product, nicotine content and burning rate could be controlled.

Example B-17

[Bifidobacterium bacteria growth promoting lactic acid drink]

10,000 parts by weight of skim milk was heat sterilized at 80 ° C. for 20 minutes, cooled to 40 ° C., and 300 parts by weight of a starter (lactic acid bacterium) was added thereto, followed by fermentation at 35 to 37 ° C. for 10 hours. Subsequently, after homogenizing, 4,000 parts by weight of isomerized sugar syrup, 2,000 parts by weight of sucrose and 170 parts by weight of powdered pullulan high content prepared by the method of Example A-1 were dissolved and maintained at 70 ° C. for sterilization. After cooling, a small amount of fragrance was added to the bottle to prepare a bifidobacterium bacteria growth promoting lactic acid beverage of the conventional lactic acid beverage type.

This product not only promotes Bifidobacterium bacteria growth but also shows the effects of plant fiber, lowers intestinal pH, inhibits the growth of decaying bacteria and prevents constipation.

Example B-18

[Low calorie bread]

As part of the main material, water was added to 6 parts by weight and 12 parts by weight of amylose, and kneaded in a mixer. 21.4 parts by weight of subsidiary materials (6 parts by weight of margarine, 2.5 parts by weight of sucrose, 6 parts by weight of eggs, 0.4 parts by weight of leaves and 6.5 parts by weight of water) and fermentation material (0.8 parts by weight of dry yeast, 0.4 parts by weight of sucrose) 5.2 parts by weight of water) was added to give a dough of suitable firmness. Subsequently, the first fermentation was carried out at 30 ° C. for 60 minutes to remove the gas generated therein. Again, the remaining 12 parts by weight of the main material (3 parts by weight of the powder and 9 parts by weight of powdered pullulan high content prepared by the method of Example A-2) were added, kneaded and mixed uniformly, and the required amount of water was added to prepare the dough. The firmness was adjusted. It left for 60 minutes in the refrigerator for 2nd fermentation. The third fermentation was carried out at 35 ° C. for 30 minutes to remove the formed gas and to form. Again, the fourth fermentation was carried out at 35 ° C. for 30 minutes and then placed in an oven at 210 ° C. and baked for 20 minutes. The control bread was used in the same manner as above except that only 30 parts by weight of the strong component was used as the main material and 8 parts by weight of water, which was a submaterial.

The appearance of the bread was darker in color and the internal structure was uniform than that of the control bread. The taste was almost free from the normal bread, which was a control, and had a slightly lighter feel as well as no smell and meaning. The attachment to the teeth was hardly felt, bite off, and the epidermis was almost firm.

It can be used not only as a low-calorie food, but also as a Bifidobacterium bacterium growth promoting effect and as a dietary fiber, which can be advantageously used for improving beauty and health maintenance.

Example B-19

[Yearless Skirt]

45.0 parts by weight of the second calcium phosphate, high liquid pullulan prepared by the method of No. 4 of Example A-3 as a 10 w / w% aqueous solution, 30.0 parts by weight, polyethylene glycol (average molecular weight 1,500) 30.0 parts by weight, 1.5 parts by weight of sodium lauryl sulfate, 17.0 parts by weight of glycerin, 0.5 parts by weight of polyoxyethylene sorbitan monolaurate, α-glycosyl stevioside sweetener (manufactured by Nippon Daiichi Co., Ltd., trade name "αG Sweet ") 0.2 parts by weight and 0.05 parts by weight of preservatives were mixed according to a conventional method to prepare a soft apron.

This product has a moderate sweetness and is suitable for children's teeth. In addition, this product contains a combination of pullulan and polyethylene glycol, and thus, it is an easy-to-use soft polish without stickiness compared to the case of using pullulan alone.

Example B-20

[Cosmetic Pack]

0.5 parts by weight of linolenic acid according to a conventional method 1.5 parts by weight of squalene, 0.5 parts by weight of polyoxyethylene cured castor oil, 5.5 parts by weight of sodium L-lactic acid, 4.0 parts by weight of glycerin, by the method of No. 2 of Example A-3 The cosmetic liquid pack was prepared by uniformly mixing the prepared liquid high content of pullulan in an aqueous solution having a concentration of 40 w / w%, 50.0 parts by weight, 10.0 parts by weight of ethyl alcohol, and 33.0 parts by weight of purified water.

This product is suitable as a whitening agent and can be advantageously used for the treatment and prevention of localized pigmentation such as brown spots, freckles, sunburn, and systemic pigmentation such as Addisonism.

Example B-21

[Shampoo]

A high liquid pullulan content prepared by the method of No. 4 of Example A-3 as a 2.5 w / w% aqueous solution, 80.0 parts by weight of ethyl alcohol 13.0 parts by weight, glycerin 2.0 parts by weight, fragrance 0.3 parts by weight, 1.5 parts by weight of polyoxyethylene sorbitan monolaurate was added and mixed to dissolve to prepare a shampoo.

This product is a shampoo that can easily wash your hair with your fingers, feels soft to the hair after washing your hair, and has an excellent feeling of use and freshness.

Example B-22

[Double Party]

A predetermined weight of 150 mg is used as the core, and 40 parts by weight of crystalline maltitol and a liquid high content of pullulan prepared by the method of No. 2 of Example A-3 are 10 w. / w% aqueous solution of 20 parts by weight, 12 parts by weight of water, 25 parts by weight of talc and 3 parts by weight of titanium oxide, using a first coating solution to the weight of the tablet up to about 230 mg, and then 65 parts by weight of crystalline maltitol, and a second coating solution of 10 parts by weight of aqueous solution of pullulan at a concentration of 10 w / w% and 25 parts by weight of water, were coated, and then coated again with a wax solution to have an excellent glossy outer surface. Dragee was prepared.

This product is easy to sugar work, has excellent impact resistance and maintains high quality for a long time.

Example B-23

[Substituted plasma]

A high liquid pullulan content prepared by the method of No. 7 of Example A-3 was used as a 10 w / w% aqueous solution, and methanol was added thereto to 40 v / v%, and the resulting lower layer was removed. Methanol was added again to 55 v / v% in the obtained upper layer, and the lower layer was fractionated. Methanol in the obtained lower layer was distilled off, activated carbon was added to the remaining aqueous solution of pullulan, decolorized, desalted with H ⁺ and OH ^- type ion exchange resins, and the purified pullulan obtained by filtration with a membrane filter was concentrated. By drying, grinding ( ) Is 1.4 and ( A pyrogen-free pullulan white powder having) of 50,000 was obtained in a yield of about 45%. Thus prepared pullulan was used as an aqueous solution of 4-10 w / v%, to which an isotonic agent such as salts and sugars was added and sterilized to prepare an injection.

This product is suitable as a plasma extender, blood flow improver, etc.

As described above, the present invention finds that higher pullulan productivity can be obtained by continuous culturing while maintaining a nutrient medium having a sugar temperature of 10 to 20 w / v% at 30 cp or less. The following pullulan high content, its manufacturing method, and use are established.

The continuous culture method of the present invention facilitates the bulk and low-cost supply of high content of pullulan, which is contained in thickeners, coating agents, adhesives, moldings, and the like, and in foods, cosmetics, pharmaceuticals, and also agricultural and forestry shrinkage materials, paper Its industrial significance is large because its use is pioneered in processing materials, mining and industrial materials.

Claims (5)

A culture obtained by continuously culturing oreovacsidium microorganism having pullulan production ability at pH 2.0 or higher and 4.0 or lower while maintaining the viscosity of the medium at 30 centipoise or less in a nutrient medium containing a sugar temperature of 10 to 20 w / v%. After concentrating, the method of producing a high pullulan content, characterized in that to recover a culture of the high concentration of pullulan. According to claim 1, wherein the sugar is selected from the group consisting of glucose, fructose, sucrose, maltose, maltooligosaccharides, isomaltooligosaccharides, starch hydrolyzate, saccharified starch, invert sugar, isomerized sugar and molasses. Manufacturing method. The production method according to claim 1, wherein the continuous culture is carried out under aerobic conditions. The method of claim 1, wherein the continuous culture is performed in the presence of amylase that partially hydrolyzes pullulan. The method of claim 4, wherein the amylase is selected from the group consisting of liquefied α-amylase and isoamylase.